Our team will evaluate the potential for gene therapy to reduce life threatening symptoms in tuberous sclerosis complex (TSC), which affects 2 million people world-wide. TSC is an autosomal dominant, tumor suppressor disorder caused by an inherited mutation in either TSC1 (encoding hamartin) or TSC2 (encoding tuberin) with somatic loss of the corresponding normal allele leading to hamartomas (focal cell overgrowths) in different organs. Neurological involvement includes cortical tubers, subependymal nodules and subependymal giant cell astrocytomas with hydrocephalus epilepsy, autism, cognitive impairment and mental health issues. We have created a stochastic central nervous system (CNS) mouse model of TSC2 which manifests subependymal overgrowths, hydrocephalus and early death, reflecting what happens in TSC patients. Current standards-of- care for subependymal lesions in the brain involve neurosurgical craniotomy and/or long term treatment with rapamycin, both having potentially damaging effects on brain development. Our thesis is that a life-threatening complication of TSC ? hydrocephalus can be prevented with a less invasive and longer lasting procedure, i.e. intravenous (IV) delivery of an adeno-associated virus (AAV) vector encoding a replacement protein, without compromising use of other standards-of-care if needed. IV delivery should achieve ?extra copies? of the replacement gene in peripheral tissues reducing the likelihood of other life threatening hamartomas forming in the body. We will use our CNS Tsc2 mouse model to evaluate the ability of an AAV vector encoding a condensed version of tuberin (cTuberin) to reduce the size of ependymal abnormalities and extend lifespan. R61 Aim 1 - Evaluation of cTuberin protein in culture, including use of induced pluripotent stem cells from patients differentiated into neural progenitor cells, with cytoxicity and functional assays, Go-no-go: cTuberin must show at least 50% of tuberin activity in suppression of mTOR activity, and normalization of cell size and rate of cell proliferation. Aim 2 - Optimize AAV serotype and promoter with AAV-cTuberin in CNS Tsc2 mouse model and evaluate therapeutic efficacy. Behavior, survival times, neuropathology and whole body pathology will be monitored, as well as vector distribution and the ability of the vector to target cells in the brain and peripheral tissues. Go-no-go: AAV-cTuberin must at least double the average lifespan in this CNS Tsc2 model and show comparable effectiveness to rapamycin (positive control). In R33 Aim 3 - Evaluate dose escalation and potential toxicity of AAV-cTuberin in Tsc2+/- and CNS Tsc2 mouse models, as well as obtaining quantitative measurements of ventricular volumes in treated and non-treated mice by magnetic resonance imaging. Our team of TSC experts includes Drs. Breakefield (preclinical gene therapy for neurologic diseases), Maguire (AAV vectors), Ramesh (biochemistry and cell biology), Stemmer-Rachamimov (neuroanatomy) and Thiele (clinical care). These activities are aimed to carry out preclinical research in support of clinical trials, to secure intellectual property rights through our institution, and to facilitate licensing to biotechnology companies.